Caffeine induces hyperacetylation of histones at the MEF2 site on the Glut4 promoter and increases MEF2A binding to the site via a CaMK-dependent mechanism

This study was conducted to explore the mechanism by which caffeine increases GLUT4 expression in C2C12 myotubes. Myoblasts were differentiated in DMEM containing 2% horse serum for 13 days and the resultant myotubes exposed to 10 mM caffeine in the presence or absence of 25 mu M KN93 or 10 mM dantrolene for 2 h. After the treatment, cells were kept in serum-free medium and harvested between 0 and 6 h later, depending on the assay. Chromatin immunoprecipitation (ChIP) assays revealed that caffeine treatment caused hyperacetylation of histone H3 at the myocyte enhancer factor 2 (MEF2) site on the Glut4 promoter (P < 0.05) and increased the amount of MEF2A that was bound to this site similar to 2.2-fold (P < 0.05) 4 h posttreatment compared with controls. These increases were accompanied by an similar to 1.8-fold rise (P < 0.05 vs. control) in GLUT4 mRNA content at 6 h post-caffeine treatment. Both immunoblot and immunocytochemical analyses showed reduced nuclear content of histone deacetylase-5 in caffeine-treated myotubes compared with controls at 0 -2 h posttreatment. Inclusion of 10 mM dantrolene in the medium to prevent the increase in cytosolic Ca2+, or 25 mu M KN93 to inhibit Ca2+ / calmodulin-dependent protein kinase (CaMK II), attenuated all the above caffeine-induced changes. These data indicate that caffeine increases GLUT4 expression by acetylating the MEF2 site to increase MEF2A binding via a mechanism that involves CaMK II.